Screening for neonatal inherited metabolic disorders by tandem mass spectrometry in Guangzhou.

To analyze the screening results for inherited metabolic disorders (IMD) in newborns by tandem mass spectrometry (MS/MS) in Guangzhou.A total of 272 117 newborns in Guangzhou from Jan 2015 to Dec 2020 were screened for IMD by MS/MS in Guangzhou Newborn Screening Center. When the primary screening was positive, the newborns and their mothers were recalled. For those with positive in re-examination, the biochemical and related genetic analysis were required for confirmation. The screening results, clinical characteristics and outcomes of the confirmed cases were retrospectively analyzed and the performance was optimized. Among 272 117 neonates, 1808 (0.66%) cases were positive in primary screening, and 1738 cases (96.13%) were recalled for review. The median clinical diagnosis time was 15 d after birth. A total of 79 cases of IMD were diagnosed, including 23 with aminoacidopathy, 17 with disorder of organic acid metabolism and 39 with fatty acid oxidation disorders, involving 21 diseases. The incidence rate was 1/3444 in newborns, and the positive predictive value of 4.5%. Four false negative cases were found, all of them were citrin deficiency. The common diseases were primary carnitine deficiency (26.6%), methylmalonic aciduria (12.7%) and phenylalanine hydroxylase deficiency (11.4%). The mothers of 32 cases were confirmed, including 30 cases of primary carnitine deficiency, 1 case of isobutyl-coenzyme A dehydrogenase deficiency and 1 case of 3-methylcromaryl coenzyme A carboxylase deficiency. The detection rate was 1/2451 in total population. During the follow-up, most patients remain asymptomatic, except for 5 severe cases who died early (1 case of maple syrup urine disease, 2 cases of isolated methylmalonic acidurmia, and 2 cases of carnitine-acylcarnitine translocase deficiency); and 10 cases of organic acid metabolism disorders developed mild psychomotor developmental retardation. After optimizing the screening indicators, the number of initial screening positives dropped to 903, and the positive predictive value increased to 9.1%, and no confirmed cases were missed. The incidence rate of fatty acid oxidation disorders is high in Guangzhou. A variety of IMD can be effectively screened out by MS/MS, and the screening performance can be improved by optimizing screening indicators.

Two novel mutations in the ALPL gene of unrelated Chinese children with Hypophosphatasia: case reports and literature review.

OBJECTIVE: Hypophosphatasia (HPP) is an inherited disorder of defective skeletal mineralization caused by mutations in the ALPL gene that encodes the Tissue Non-specific Alkaline Phosphatase (TNSALP). It is subdivided into six forms depending on the age of onset: perinatal lethal, prenatal benign, infantile, childhood, adult, and odonto HPP. Among these, infantile HPP is characterized by early onset and high frequency of lethal outcome. Few studies have reported the phenotype and genetic characteristics of HPP in Chinese children. CASE PRESENTATION: Three forms of HPP were identified in four unrelated patients from four different Chinese families, including one lethal infantile (patient 1), two childhood (patient 2 and 3) and one odonto HPP (patient 4). Six variants in the ALPL gene were identified, including five missense mutations and one frameshift mutation. Of which, none were reported previously in the Chinese population, and two were novel (c.359G > C: p.G120A and c.1017dupG: p.H340AfsX3). Patient 1 carrying a novel homozygous (c.359G > C) mutation showed respiratory distress and pneumonia at first day of his life. He presented nearly negligible level of serum ALP activity, overall skeletal hypominaralization and died at 3 months old. Patient 2, 3 and 4 were compound heterozygotes with decreased serum ALP activity. Patient 2 and 3 presented premature loss of deciduous teeth, muscle weakness and bone pain, whereas patient 4 had early loss of deciduous teeth only. All four pedigrees exhibited autosomal recessive pattern of inheritance. CONCLUSIONS: In this study, six mutations in the ALPL gene were found in four Chinese HPP patients, two of which were novel: c.359G > C in exon 5 and c.1017dupG in exon 10. Our results strongly indicated that the novel mutation c.359G > C might be disease-causing and associated with severe infantile form of HPP.

Target of rapamycin complex 1 and Tap42-associated phosphatases are required for sensing changes in nitrogen conditions in the yeast Saccharomyces cerevisiae.

In yeast target of rapamycin complex 1 (TORC1) and Tap42-associated phosphatases regulate expression of genes involved in nitrogen limitation response and the nitrogen discrimination pathway. However, it remains unclear whether TORC1 and the phosphatases are required for sensing nitrogen conditions. Utilizing temperature sensitive mutants of tor2 and tap42, we examined the role of TORC1 and Tap42 in nuclear entry of Gln3, a key transcription factor in yeast nitrogen metabolism, in response to changes in nitrogen conditions. Our data show that TORC1 is essential for Gln3 nuclear entry upon nitrogen limitation and downshift in nitrogen quality. However, Tap42-associated phosphatases are required only under nitrogen limitation condition. In cells grown in poor nitrogen medium, the nitrogen permease reactivator kinase (Npr1) inhibits TORC1 activity and alters its association with Tap42, rendering Tap42-associated phosphatases unresponsive to nitrogen limitation. These findings demonstrate a direct role for TORC1 and Tap42-associated phosphatases in sensing nitrogen conditions and unveil an Npr1-dependent mechanism that controls TORC1 and the phosphatases in response to changes in nitrogen quality.

A pilot study of newborn screening for X-linked adrenoleukodystrophy based on liquid chromatography-tandem mass spectrometry method for detection of C26:0-lysophosphatidylcholine in dried blood spots: Results from 43,653 newborns in a southern Chinese population.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a rare X-linked disease caused by mutations of the ABCD1 gene. C26:0-lysophosphatidylcholine (C26:0-LPC) has been proved to be an accurate biomarker for X-ALD. This study aims to propose an effective method for screening of X-ALD and to evaluate the performance of the newborn screening (NBS) assay for X-ALD in Guangzhou. METHODS: C26:0-LPC in dried blood spots (DBS) was extracted by methanol solution containing isotope-labelled internal standard (C26:0-d4-LPC) and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sensitivity of the method was assessed in eight male X-ALD patients, two female carriers and 583 healthy controls. The method was conducted on 43,653 newborns. Next generation sequencing was performed on screen-positive samples. Plasma analysis of very long-chain fatty acids and genetic counselling were performed by way of follow-up. RESULTS: Elevated C26:0-LPC were 100% sensitive for screening of X-ALD. Of 43,653 newborns, 32 (18 males, 14 females) screened positive. Of these, 14 (43.7%) were identified ABCD1 variants, including seven hemizygous males and seven heterozygous females, and two (6.3%) were diagnosed with other peroxisomal disorders. CONCLUSION: The LC-MS/MS method for screening of X-ALD can identify males, heterozygous females and other peroxisomal disorders. The incidence of X-ALD in Guangzhou is not low.

AAV9-coGLB1 Improves Lysosomal Storage and Rescues Central Nervous System Inflammation in a Mutant Mouse Model of GM1 Gangliosidosis.

BACKGROUND: GM1 gangliosidosis (GM1) is an autosomal recessive disorder characterized by the deficiency of beta-galactosidase (ß-gal), a ubiquitous lysosomal enzyme that catalyzes the hydrolysis of GM1 ganglioside. OBJECTIVE: The study aims to explore the application of the AAV9-coGLB1 for effective treatment in a GM1 gangliosidosis mutant mouse model. METHODS: We designed a novel adeno-associated virus 9 (AAV9) vector expressing ß-gal (AAV9- coGLB1) to treat GM1 gangliosidosis. The vector, injected via the caudal vein at 4 weeks of age, drove the widespread and sustained expression of ß-gal for up to 32 weeks in the Glb1G455R/G455R mutant mice (GM1 mice). RESULTS: The increased levels of ß-gal reduced the pathological damage occurring in GM1 mice. Histological analyses showed that myelin deficits and neuron-specific pathology were reduced in the cerebral cortex region of AAV9-coGLB1-treated mice. Immunohistochemical staining showed that the accumulation of GM1 ganglioside was also reduced after gene therapy. The reduction of the storage in these regions was accompanied by a decrease in activated microglia. In addition, AAV9 treatment reversed the blockade of autophagic flux in GM1 mice. CONCLUSION: These results show that AAV9-coGLB1 reduces the pathological signs of GM1 gangliosidosis in a mouse model.

A GM1 gangliosidosis mutant mouse model exhibits activated microglia and disturbed autophagy.

GM1 gangliosidosis is a rare lysosomal storage disease caused by a deficiency of ß-galactosidase due to mutations in the GLB1 gene. We established a C57BL/6 mouse model with Glb1G455R mutation using CRISPR/Cas9 genome editing. The ß-galactosidase enzyme activity of Glb1G455R mice measured by fluorometric assay was negligible throughout the whole body. Mutant mice displayed no marked phenotype at eight weeks. After 16 weeks, GM1 ganglioside accumulation in the brain of mutant mice was observed by immunohistochemical staining. Meanwhile, a declining performance in behavioral tests was observed among mutant mice from 16 to 32 weeks. As the disease progressed, the neurological symptoms of mutant mice worsened, and they then succumbed to the disease by 47 weeks of age. We also observed microglia activation and proliferation in the cerebral cortex of mutant mice at 16 and 32 weeks. In these activated microglia, the level of autophagy regulator LC3 was up-regulated but the mRNA level of LC3 was normal. In conclusion, we developed a novel murine model that mimicked the chronic phenotype of human GM1. This Glb1G455R murine model is a practical in vivo model for studying the pathogenesis of GM1 gangliosidosis and exploring potential therapies.

Detection of glucosylsphingosine in dried blood spots for diagnosis of Gaucher disease by LC-MS/MS.

INTRODUCTION: Gaucher disease (GD) is caused by a deficiency of ß-glucosidase (GCase), leading to accumulation of glucosylceramide (GlcC) and glucosylsphingosine (Lyso-Gb1). Lyso-Gb1 is a reliable biomarker for GD. OBJECTIVES: This study aims to develop a simple, effective and accurate method for the screening and diagnosis of GD using dried blood spot (DBS) samples. METHODS: Lyso-Gb1 in DBS was extracted by 50% acetonitrile aqueous solution containing isotope-labeled internal standard and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A reference interval was established by analyzing samples from 277 healthy controls. Lyso-Gb1 was detected in the residual DBS samples from 142 high-risk patients with splenomegaly and/or thrombocytopenia. Based on GCase activity in DBS, samples were classified into four groups: confirmed GD patients (n = 52), GD carriers (n = 5), false positive (n = 36) and negative (n = 49). RESULTS: The optimized Lyso-Gb1 assay showed intra- and inter-assay variations ranged between 2.0%-8.2% and 3.8%-10.2%, respectively. Accuracies ranged from 93.5% to 112.6%. The lowest limit of quantification was 1 ng/mL. The normal reference interval of Lyso-Gb1 in DBS ranged from 2.1 to 9.9 ng/mL. Among the 142 subjects, except for one GD patient (Lyso-Gb1 > 2500 ng/mL), the Lyso-Gb1 concentrations in 51 GD patients ranged from 190.5 to 2380.6 ng/mL (the median 614.8 ng/mL). Also, one negative patient was found to have an elevated Lyso-Gb1 level (684.5 ng/mL), while the other patients were normal. The negative case was then confirmed to be an atypical GD patient with a c.1091A > G (p.Y364C) homozygous variant in PSAP gene by next generation sequencing. CONCLUSIONS: The optimized method to determine Lyso-Gb1 in DBS was demonstrated as a useful tool for the screening and diagnosis of GD.

High risk screening for Gaucher disease in patients with splenomegaly and/or thrombocytopenia in China: 55 cases identified.

Gaucher disease (GD) is a common lysosomal storage disorder caused by deficiency of glucocerebrosidase (GCase) due to the pathogenic variants in the GBA gene. The aim of this study was to evaluate the performance of high risk screening program for GD by measuring the enzyme activities of GCase and chitotriosidease in dried blood spots of patients with splenomegaly and/or thrombocytopenia. A total of 787 subjects (364 females and 423 males) with unexplained splenomegaly and/or thrombocytopenia were enrolled in this study from May 2016 to Aug 2019. The cutoff value of GCase activity was set as less than 3.0 pmol/punch/h for screening positive. The diagnosis of GD was confirmed by Sanger sequencing of the GBA gene. Among 131 screening positive cases, 49 patients were confirmed GD. The positive predictive value was 37.4%.Three patients with boundary values (GCase 3-4 pmol/punch/h) and other three splenectomic patients with normal GCase activity were confirmed GD by GBA genetic analysis because of increased chitotriosidase or Gaucher cells in bone marrow. A total of 55 GD cases were identified. The sensitivity and specificity of the high risk screening were 98.2% and 89.5%, respectively. These 55 GD patients presented splenomegaly (100%), hepatomegaly (70.9%), thrombocytopenia (83.6%). The level of GCase in GD patients was (1.7 ± 1.6) pmol/punch/h. The increased chitotriosidase (383.8 ± 130.2 pmol/punch/h) was found in 42 (76.4%) patients with GD. Molecular genetic analysis identified 44 variants in the GBA gene, including 11 novel variants. The results showed the high risk screening for GD is accurate, rapid and cost-effective.

Same-sex twins have a high incidence of congenital hypothyroidism and a high probability to be missed at newborn screening.

BACKGROUND: We estimated the incidence of CH in twins, analyse the clinical features of CH cases in twins and further evaluate the CH screening strategy and recall procedures for twins. METHODS: A retrospective investigation of the screening results and confirmed cases in 724,791 newborns was conducted from 2015 to 2017 in Guangzhou. Clinical features were compared between twins with CH and singletons with CH. In addition, the twins were further divided into same-sex twins and different-sex twins to analyse the characteristics and incidence of CH and to compare differences in the confirmed cases in the 2 groups. RESULTS: The incidence of CH in same-sex twins was 1/593, which was much higher than the incidence of CH in singletons (1/1323) and different-sex twins (1/3060). Of the 20 twins diagnosed with CH, 17 were same-sex twins and 3 were different-sex twins. Among the six pairs of same-sex twins with CH, four had TSH inconsistency, which reached 67%. Eight of the 17 cases of same-sex twins diagnosed with CH had negative results at the first screening. CONCLUSIONS: Distinguishing same-sex twins from different-sex twins during newborn screening is more feasible. The incidence of CH in same-sex twins is much higher than that in the general population and the risk of transient CH is relatively high. In positive cases in same-sex twins, the simultaneous recall of the twin can effectively avoid a missed diagnosis. The screening center should properly evaluate the recall strategy and screening procedure for twins, especially twins of the same-sex.

The adjustment of 17-hydroxyprogesterone cut-off values for congenital adrenal hyperplasia neonatal screening by GSP according to gestational age and age at sampling.

Background Congenital adrenal hyperplasia (CAH) screening is facing great challenges because of a high false-positive rate and a low positive predictive value (PPV). We established and optimized 17-hydroxyprogesterone (17-OHP) cut-off values for CAH neonatal screening using a genetic screening processor (GSP) according to gestational age (GA), birth weight (BW) and age at sampling. Methods The 17-OHP concentrations in dried blood spots were measured by time-resolved immunofluorescence and were grouped in terms of GA, BW and age at sampling for 48,592 newborns. The 99.5th percentile was used to set an initial cut-off value as a reference. Results Significant differences in 17-OHP concentrations were observed among newborns with different GAs and BWs. A significant difference was observed among different sampling age groups. Finally, we defined new multitier cut-off concentrations based on GA and age at sampling. Application of the new cut-off values resulted in a 30% reduction of the positive rate and a 40% increase of the PPV. Conclusions GA, BW and sampling age time influenced the concentrations of 17-OHP. The efficiency of congenital adrenal hyperplasia screening can be substantially improved by adjusting the multitier cut-off value according to GA and age at sampling.

Identification of an inherited pathogenic DNAJC12 variant in a patient with hyperphenylalalinemia.

Hyperphenylalaninemia (HPA), an abnormal condition of phenylalanine metabolism, was recently reported to be caused by DNAJC12 mutations. As the heat shock co-chaperone, DNAJC12 prevents the aggregation of misfolded or aggregation-prone proteins and maintain the correct assembly and degradation. Here, we report a patient with unexplained HPA detected by newborn screening. Differential diagnoses of pterin profile and targeted next generation sequencing of excluded the most common causes of the defects of the enzyme phenylalanine hydroxylase or its cofactor tetrahydrobiopterin (BH4). Sanger sequencing revealed a novel homozygous deletion variant of c.262del in DNAJC12, which was predicted to produce the truncated protein (p.Q88SfsTer6) and was considered pathogenic to result in the symptoms of global developmental delays clinically. Treatment with the combination of BH4, the neurotransmitter precursors of dopamine and serotonin, and phenylalanine-restricted diet enabled the patient to improve his development and stabilize his phenylalanine level in a reasonable range. These findings expanded the spectrum of the DNAJC12 mutations and provided new insights on patient management, further supporting the causal relationships of DNAJC12 and HPA.

Clinical and molecular characteristics of carnitine-acylcarnitine translocase deficiency: Experience with six patients in Guangdong China.

Carnitine-acylcarnitine translocase deficiency (CACTD) is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation that occurs due to mutations in the SLC25A20 gene. Severe CACTD results in neonatal or infantile sudden death. Herein, we reported six patients with CACTD diagnosed based on biochemical and molecular findings from 5 unrelated families in Guangdong from 2016 to 2017. Among them, five patients presented with hypotonia, nonketotic hypoglycemia, and arrhythmia 2 days after birth, while the other patient presented with respiratory distress, hypotonia, and arrhythmia. Five of the patients died in the neonatal period. Blood acylcarnitine concentrations determination from dried blood spots (DBS) were measured by tandem mass spectrometry (MS/MS). The SLC25A20 and CPT2 gene sequences were analyzed by direct Sanger sequencing. SLC25A20 gene analysis revealed a c.199-10T>G (IVS2-10T>G) homozygous variants in four unrelated patients and a novel mutation c.199-10T>G/c.719-8_c.719-1dupCCCACAG compound heterozygous variants in twins. This report describes the clinical characteristics, biochemical findings and molecular analysis of SLC25A20 gene of patients with CACTD in Guangdong. And our results show that the c.199-10T>G is likely the most common variant of CACTD in Guangdong population as it accounts for 83% (10/12) of the observed mutant alleles. Individuals with the c.199-10T>G genotype had a severe CACTD phenotype.

Loss of Rictor with aging in osteoblasts promotes age-related bone loss.

Osteoblast dysfunction is a major cause of age-related bone loss, but the mechanisms underlying changes in osteoblast function with aging are poorly understood. This study demonstrates that osteoblasts in aged mice exhibit markedly impaired adhesion to the bone formation surface and reduced mineralization in vivo and in vitro. Rictor, a specific component of the mechanistic target of rapamycin complex 2 (mTORC2) that controls cytoskeletal organization and cell survival, is downregulated with aging in osteoblasts. Mechanistically, we found that an increased level of reactive oxygen species with aging stimulates the expression of miR-218, which directly targets Rictor and reduces osteoblast bone surface adhesion and survival, resulting in a decreased number of functional osteoblasts and accelerated bone loss in aged mice. Our findings reveal a novel functional pathway important for age-related bone loss and support for miR-218 and Rictor as potential targets for therapeutic intervention for age-related osteoporosis treatment.

Osteoblasts secrete Cxcl9 to regulate angiogenesis in bone.

Communication between osteoblasts and endothelial cells (ECs) is essential for bone turnover, but the molecular mechanisms of such communication are not well defined. Here we identify Cxcl9 as an angiostatic factor secreted by osteoblasts in the bone marrow microenvironment. We show that Cxcl9 produced by osteoblasts interacts with vascular endothelial growth factor and prevents its binding to ECs and osteoblasts, thus abrogating angiogenesis and osteogenesis both in mouse bone and in vitro. The mechanistic target of rapamycin complex 1 activates Cxcl9 expression by transcriptional upregulation of STAT1 and increases binding of STAT1 to the Cxcl9 promoter in osteoblasts. These findings reveal the essential role of osteoblast-produced Cxcl9 in angiogenesis and osteogenesis in bone, and Cxcl9 can be targeted to elevate bone angiogenesis and prevent bone loss-related diseases.